Protocol for genome-wide CRISPR knockout screens of bacterial cytotoxins in HeLa cells

Summary CRISPR screening is a powerful tool to identify host factors for pathogenic agents including viruses and bacterial toxins. Here, we present a protocol to conduct a genome-scale CRISPR screen on HeLa cells for host factors involved in the toxin action of Clostridioides difficile TcdB4. We describe in detail how to prepare the library, set up the screen, obtain the gene sequences, and analyze the results. This protocol can also be modified for other genome-scale libraries, cell lines, and cytotoxins. For complete details on the use and execution of this protocol, please refer to Luo et al. (2022).

*Correspondence: taoliang@westlake.edu.cn https://doi.org/10. 1016/j.xpro.2022.101595 SUMMARY CRISPR screening is a powerful tool to identify host factors for pathogenic agents including viruses and bacterial toxins. Here, we present a protocol to conduct a genome-scale CRISPR screen on HeLa cells for host factors involved in the toxin action of Clostridioides difficile TcdB4. We describe in detail how to prepare the library, set up the screen, obtain the gene sequences, and analyze the results. This protocol can also be modified for other genome-scale libraries, cell lines, and cytotoxins. For complete details on the use and execution of this protocol, please refer to Luo et al. (2022).

BEFORE YOU BEGIN
The protocol below describes the CRISPR knockout screen for TcdB4 on HeLa cells using the GeCKO v2 library. However, we have also used this protocol in other human cell lines and with other genome-wide CRISPR libraries. To minimize possible biases, we strongly suggest using one batch of the toxin throughout the screen. Bacterial toxins can be divided into small aliquots and stored at À80 C for one year without overt loss of activity. The toxin should be used immediately after being thawed, and never refreeze and/or reused thawed toxin aliquots. Earlier versions of this protocol were applied for targeted screens with other bacterial cytotoxins (Tao et al., 2016(Tao et al., , 2019Zhou et al., 2021). For complete details on the use and execution of the protocol, please refer to (Luo et al., 2022).

Model cell line determination
Timing: 3 days Below we provided a detailed step-by-step protocol for testing the sensitivity of multiple cell lines to the target toxin (here TcdB4). The determined cell line is adopted for the CRISPR screen for TcdB4.
1. Grow candidate cell lines. After the trypsin digestion, collect and dilute the cells to appropriate concentrations with the respective medium. Seed each cell line to a 24-well plate and allow them to grow to a $60% confluent before adding the toxin. The inoculating density for each cell line may be different.

Note:
The choice of the model cell line is critical to performing the CRISPR screen, as resistant cells possibly lack the host factors for the toxin actions. In this protocol, we tested five cell lines including HeLa, U2OS, A549, MCF-7, and HT-29.
2. Prepare serial dilutions of the toxin (here TcdB4) within DMEM complete (DMEM supplemented with 10% FBS, 50 units/mL of penicillin, and 50 mg/mL of streptomycin). Dilute the stocked TcdB4 to 1 mM as a starting concentration. 3. Perform the serial dilution with a dilution factor of three. Prepare a total of 23 dilutions and final concentrations of TcdB are the following: 1 mM, 0.33 mM, 0.11 mM, 0.037 mM, 0.012 mM, 4 nM, 1.4 nM, 0.46 nM, 0.15 nM, 0.051 nM, 0.017 nM, 5.6 pM, 1.9 pM, 0.63 pM, 0.21 pM, 0.070 pM, 0.023 pM, 0.0077 pM, 0.0026 pM, 0.00086 pM, 0.00029 pM, and 0.0001 pM. 4. Remove the spent medium of the cell culture in the 24-well plate and add 0.5 mL of toxin dilution to each well. Leave one well of cells with the fresh medium as an untreated control. Put the plates back into the cell culture incubator. 5. After 14 h, capture the bright-field images of each well under the microscope. Manually count the numbers of round-shaped and normal-shaped cells ( Figure 1A), calculate the cell rounding ratio, and plot them onto the toxin dose-cell rounding curves ( Figure 1B). 6. Calculate the CR 50 (toxin concentration that induces 50% of cell rounding) from the cell rounding curves for each cell line ( Figure 1B). Cell lines with low CR 50 value would be ideal model cells for the CRISPR screen. Here, HeLa cells are sensitive to TcdB4 and thus were chosen for the screen.
Generating HeLa-Cas9 cell line Timing: 4-6 weeks We utilize lentivirus to integrate the Streptococcus pyogenes cas9 gene together with the blasticidin resistance gene into the genome of HeLa cells.

OPEN ACCESS
7. Preparing Cas9 lentivirus. a. Seed 2 3 10 6 HEK293T cells into a 10 cm dish with the antibiotic-free DMEM complete. Grow the cells at 37 C with 5% CO 2 . The cells should achieve 80% confluency after 24 h of incubation. b. The transfection using PolyJet transfection reagent is performed following the manufacturer's protocol (https://signagen.com/DataSheet/SL100688.pdf). The amounts of transfection reagent and plasmids are used as follows: set up a mixture of plasmids: 0.25 mL DMEM (serum-free), 3 mg psPAX2, 0.3 mg pMD2.G, and 3 mg pLenti-cas9-blasticidin; set up a transfection agent mixture: 0.25 mL DMEM (serum-free) and 18 mL of Polyjet. c. At 18 h post-transfection, replace the medium with 20 mL of fresh DMEM complete. Grow the cells for additional 30 h at 37 C in 5% CO 2 .
Note: At this step, all operations should be as gentle as possible, as 293T cells may easily be detached from the plate.
d. At 48 h post-transfection, collect the culture medium containing lentiviruses to a 15 mL sterile centrifuge tube (low retention).
Optional: To completely remove the detached cells and cell debris, centrifuge the tube at 500 g for 5 min. Then filter the supernatant through a 0.45 mm syringe filter to a new 15 mL centrifuge tube.
e. Split the lentiviral solution into 1 mL aliquots in the 1.5 mL microcentrifuge tubes. Store the lentivirus in the À80 C freezer.
CRITICAL: When working with lentivirus, specific safety procedures must be followed. All materials including cell cultures, cell culture materials, and virus stocks that have contacted lentivirus must be bleached for 15 min before disposal.
8. Lentivirus infection. a. Seed the HeLa cells at a density of 5 3 10 5 cells/mL onto a 6-well plate and incubate at 37 C in 5% CO 2 . The cells should achieve $80% confluency after 24 h of incubation. b. Dilute the lentiviral stock to the DMEM complete with dilution factors of 2, 6, 18, 54, and 162.
Add the diluted lentivirus into the cell culture dropwise and gently rock the plate three times. Leave one well of the cells without lentivirus infection as an uninfected control. Place the culture plate back in the incubator. c. At 24 h post-infection, replace the culture medium with fresh DMEM complete containing 5 mg/mL Blasticidin. d. At 72 h post-infection, remove the culture medium and wash the cells with PBS three times.
Add 2 mL of fresh DMEM complete with 2.5 mg/mL Blasticidin and continue the cultivation for additional 48 h. At this point, you should find that all cells in the uninfected control well are dead. For wells with infected cells normally grow under Blasticidin selection, pick the one with the highest viral dilution factor for following single colony isolation. 9. Single colony isolation and validation.
a. Use trypsin to digest the cells, count the cell number, and dilute to a density of 3 cells/mL in the DMEM complete. Transfer 200 mL of diluted cells into a 96-well plate. Put the plate back in the cell culture incubator. b. Check 96-well plate under a microscope after one week. Mark the wells with only one colony formed, and replace the DMEM complete media. c. Allow the cells to grow for another week and then passage colonies to the 6-well plates. Pick up at least 10 colonies. d. Once the cells reach $80% confluency, freeze stock the cells of each colony as backups, and passage the cells to new 6-well plates for validation.

OPEN ACCESS
STAR Protocols 3, 101595, September 16, 2022 e. Use RIPA to lyse the cells for each colony, load the lysate onto an SDS-PAGE, transfer it to a nitrocellulose membrane, and blot it with an anti-Cas9 antibody. The uninfected HeLa cells serve as the negative control ( Figure 2). Save the colonies with robust Cas9 expression and select one for the following study.

Estimating CR 95 for TcdB4 on HeLa-Cas9
Timing: 3 days Below we provided a detailed step-by-step protocol for estimating the CR 95 for TcdB4 on HeLa cells. The CR 95 value would be a reference toxin concentration when setting up the formal CRISPR screen.
10. Seed the HeLa-Cas9 cells with a density of 7 3 10 4 to a 24-well plate and incubate at 37 C with 5% CO 2 for 24 h. The cells should reach $60% confluency. 11. Thaw one vial of TcdB4 stock, dilute the toxin with DMEM complete to 30 pM as a starting concentration, and perform the serial dilution with a dilution factor of 3. Twelve dilutions are prepared. 12. Remove the spent medium and add 0.5 mL of toxin dilution to each well. Put the plates back into the cell culture incubator. 13. After 14 h, capture bright-field images for each well under the microscope. Count the numbers of round-shaped and normal-shaped cells, calculate the cell rounding ratio, plot them onto a toxin concentration-cell rounding chart, and align the cell rounding curve. 14. Calculate the CR 95 from the cell rounding curve for HeLa-Cas9 ( Figure 1B). This CR 95 value would be a reference concentration on the screen.

STEP-BY-STEP METHOD DETAILS
Generating human GeCKO v2 lentiviral library

Timing: 6 days
Below we provided a detailed step-by-step protocol for generating the human GeCKO v2 lentiviral library from the purchased human GeCKO v2 plasmid library.
Note: GeCKO v2 library is delivered and amplified as two half-libraries (A and B). Combine the two plasmid half-libraries with a 1:1 ratio before making the lentiviral library.
2. Seed 293T cells into two 150 mm dishes at a seeding density of 5 3 10 6 cells/plate with the antibiotic-free DMEM complete (no Penicillin/Streptomycin). 3. Grow the cells at 37 C with 5% CO 2 and allow the cells to achieve 80% confluency. 4. Transfection of HEK293T cells using PolyJet transfection reagent is performed following the manufacturer's protocol (https://signagen.com/DataSheet/SL100688.pdf) with some modifications. a. For each 150 mm plate, replace the medium with 20 mL of fresh antibiotic-free DMEM complete 30 min before transfection. b. For each 150 mm plate, prepare two clean 1.5-mL microcentrifuge tubes. Add 0.5 mL of DMEM (serum-free), 10 mg of psPAX2, 1 mg of pMD2.G, and 10 mg of GeCKO v2 plasmid library into one tube. Add 0.5 mL of DMEM (serum-free) and 48 mL of PolyJet into the other tube. Mix well by gentle pipetting respectively. c. Add the diluted PolyJet immediately into the diluted plasmids, pipette up and down 3-4 times, or vortex briefly to mix. d. Incubate the mix at 25 C for 10-15 min. Note: At this step, all operations should be as gentle as possible, as 293T cells may easily be detached from the plate.
b. At 48 h post-transfection, collect the culture medium containing the lentiviral library to a 50 mL sterile centrifuge tube (low retention).
Optional: To completely remove the detached cells and cell debris, centrifuge the tube at 500 g for 5 min. Then filter the supernatant through a 0.45 mm syringe filter to a new 50 mL centrifuge tube.
c. Split the lentiviral solution into 1 mL aliquots in the 1.5 mL microcentrifuge tubes. Store the lentivirus in the À80 C freezer.

Lentiviral titer estimation
Timing: 5 days Below we provided a detailed step-by-step protocol for estimating the titer of the lentiviral library. A lentiviral library with high titer would be beneficial to generate a high-quality pooled cell library.
6. Seed HeLa-Cas9 cells into the 6-well plate with a seeding density of 0.3 3 10 6 cells/plate and incubate the cells at 37 C with 5% CO 2 . 7. After 24 h, the cells should be $80% confluent. Thaw the GeCKO v2 lentiviral library on ice. 8. Prepare six 15 mL tubes and dilute the lentiviral library in DMEM complete with a 3-fold serial dilution (dilution factors of 3, 9, 27, 81, and 243, respectively). Leave one well without lentiviral infection as a control. 9. Remove the medium in the 6-well plate and transfer the diluted lentiviral library solution to each well. 10. After 12 h, replace the medium with fresh DMEM complete. 11. At 24 h post-transfection, change the medium with DMEM complete containing 2.5 mg/mL puromycin. 12. Incubate the cells with DMEM complete containing 2.5 mg/mL puromycin for 48 h. Trypsin digest and count live cell numbers with trypan blue staining. 13. Plot infected cell ratio (estimated by colony-forming method) versus dilution rate into a chart ( Figure 3). The multiplicity of infection (MOI) is approximately equal to the infected cell ratio at low numbers (<20%). 14. Estimate the lentivirus volume that can achieve an MOI of 0.3 for the next step (Shalem et al., , 2015.

Pooled cell library preparation
Timing: 8 days Below we provided a detailed step-by-step protocol for the preparation of the pooled CRISPR cell library. We recommend checking the gRNA coverage of the starting cell library sample before starting the toxin screen, though it can also be performed by the end of the whole screen process.
15. Seed HeLa-Cas9 cells into four 15 cm dishes at a seeding density of 8 3 10 6 cells/plate and incubate at 37 C in 5% CO 2 . 16. After 24 h of incubation, the cells would reach 80% confluent. 17. Thaw the GeCKO v2 lentiviral library on ice. Transfect the HeLa-Cas9 cells at an MOI of 0.3 and put the cells back in the incubator.
Note: Add the lentiviruses dropwise into the cell culture, and rock gently three to four times to ensure the even separation of the viruses. PCR amplification of DNA sequences encoding small guide RNA (gRNA)

Timing: 3 days
Below we provided a detailed step-by-step protocol for obtaining the pooled gDNA fragments from the collected cell library samples.

34.
A HeLa cell contains about 6 pg of genomic DNA. Each HeLa cell in the cell library is expected to harbor only one gRNA encoding gene. To ensure 20-fold coverage of the gRNA library (GeCKO v2 library contains over 100,000 gRNAs), 12 mg of genomic DNA is needed for the PCR amplification (6 pg of genomic DNA per cell X 100,000 gRNAs X 20-fold coverage).
Note: R1, R2, and R3 are expected to contain much fewer numbers of gRNAs, a lower amount of genomic DNA may be used as the template for PCR recovering gRNAs encoding sequences from these samples.
35. For PCR amplification of DNA sequences encoding gRNAs, set up PCR reactions like the following: 36. Run PCR reaction using the following conditions: 37. Agarose gel electrophoresis. Prepare a 2% agarose gel. Load 200 mL of PCR product for gRNA and run the gel. 38. Agarose gel extraction. Visualize the gel under Transilluminator ( Figure 5). Cut the $200 bp band and purify DNA using an agarose gel extraction kit. 39. Quantify DNA and send for next-generation sequencing. 100 mg DNA yield is sufficient to perform the next-generation sequencing (NGS).

NGS and data processing
Timing: 1 week In this step, we perform NGS on Illumina Novaseq 6000 platform, align raw data to human GeCKO v2 library reference, use Cutadapt (Martin, 2011) to trim down raw sequencing reads and get the count number of each gRNA.

PCR reaction master mix
f. Use the script combine_a_b.pl to combine counts from A-and B-half-libraries via typing the command: 43. The number of unique gRNAs and NGS reads for each gene are plotted in a chart.
Note: Quality control of the initial CRISPR knockout cell library (R0) is very important. In a highquality cell library, the gRNA/gene coverage should be as high as possible, while the number of NGS reads per gRNA need to be even. An example of gRNA/gene coverage in R0 is shown here (Figure 6).

EXPECTED OUTCOMES
Using this genome-wide CRISPR screen protocol in HeLa-Cas9 cells, we can uncover several host cell genes involving major steps in the TcdB4 toxin action, including cell surface recognition, endocytosis, acidification of the endosome, and production of the co-substrate UDP-glucose. An example of outcomes after three rounds of TcdB4 selection is shown in Figure 7. We have successfully unveiled two TcdB4 receptors, one enzyme responsible for producing UDP-glucose, and multiple receptor-related genes (Figure 7). For detailed outcomes, please refer to the original article (Luo et al., 2022).

LIMITATIONS
This protocol provides detailed materials and reagents for the loss-of-function screen on HeLa cells and can be modified to other cell lines or cytotoxins. We chose HeLa cells in this protocol because HeLa cells are sensitive to TcdB4 in the cell rounding assay. But this protocol can be modified and applied to other cell lines. As a loss-of-function CRISPR screen, only functional but non-essential genes expressed in the model cells will be targeted. Also, gene redundancy could reduce the screening efficiency, therefore genes with multiple homologs may not be easy (but still possible) to be targeted.

TROUBLESHOOTING Problem 1
Lentivirus integration efficiency on HeLa-Cas9 cells is much lower than the WT HeLa cells (before you begin step 8).

Potential solution
When making the HeLa-Cas9 stable cell line, avoid infecting cells with high MOI.

Problem 2
The screen results show a bias when using the resuscitated cells as starting cell library (step 22).

Potential solution
Always perform the quality control for the starting library (R0). If the gDNA coverage is low in R0, regenerate the starting cell library. Problem 3 The numbers of cytopathic cells induced by the toxin are not consistent when the assays are performed in the 12-well plate and 15-cm dish (step 26).

Potential solution
Usually, toxin concentration tested in a 12-well plate can be used directly in a 15-cm dish screen. However, if the cell rounding rate in a 15-cm dish appears different from the 12-well plate test, you can adjust toxin concentration. For instance, if very few cells survive in a given toxin concentration, try lowering the toxin concentration by 2-fold.

Problem 4
Cells get contaminated by germs during the long-term screening (step 32).

Potential solution
Sterilizing all reagents and materials before use. In particular, sterilizing the culture medium after adding the bacterial toxins by filtration through a 0.22 mm pore.
Problem 5 NGS shows that the gRNA coverage in R0 is lower than expected (step 43).

Potential solution
Don't use viruses of low titer for R0 cell library preparation.
Optimized virus package conditions as the following: Use 293T cells with low passage numbers. Estimate transient transfection efficiency with a reporter plasmid before making the library. If plasmid transfection efficiency is low, transfect using high-quality DNA and adjust the transfection reagent-DNA ratio.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Liang Tao (taoliang@westlake.edu.cn).

Materials availability
All unique/stable reagents generated in this study are available from the lead contact with a Material Transfer Agreement.